The first three enzymatic steps in the de novo synthesis of pyrimidines (carbamyl phosphate synthetase Il, aspartate transcarbamylase and dihydroorotase) are the same in all organisms, whether prokaryotic or eukaryotic. However, the structure, organization and regulation of these three enzymatic activities and their respective genes vary among species. For example, in bacteria, each enzymatic activity is associated with a separate protein. In mammals, they are found together in a single translational product of 240,000 daltons called CAD. The long term goals are to learn how having these three enzymes in one protein benefits mammals, how the de novo synthesis of pyrimidines and its regulation contribute to normal development, cellular proliferation and homeostasis, and what new agents can be developed to block this pathway in cancer cells. In order to learn more about the structure, function and regulation of CAD and its enzymatic domains, the general approach is to introduce expression vectors carrying hamster CAD cDNA, whole or in pieces, into bacteria, hamster cell lines and Drosophila defective in one or more of these enzymes. By modifying the CAD sequence through site-directed mutagenesis it becomes possible to probe the structure and active site of the aspartate transcarbamylase domain and the phosphorylation site that affects the allosteric regulation of the carbamyl phosphate synthetase domain. In situ hybridization and biochemical methods will be used to determine the pattern of CAD mRNA expression in developing mouse embryos and adult tissues, revealing where the de novo pathway contributes to cellular proliferation and where this pathway may contribute uridine to other tissues. Finally, molecular techniques will be applied with a hamster cell culture system to determine whether the 3' end of the CAD gene plays a role in its posttranscriptional regulation. By using a combination of genetic, molecular biological and biochemical approaches to study CAD, much may be learned about the normal and abnormal function of a multifunctional protein that plays a key role in an essential biosynthetic pathway.